This program investigates the role of protein-protein interactions in natural product modular synthases, a group of biosynthetic enzymes of three types: polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), and fatty acid synthase (FAS). Many of these natural products serve as anti-cancer agents or antibiotics, while others are pathogenic toxins. Recent evidence indicates that protein-protein interactions between the carrier protein domain and catalytic domains of these pathways are key to proper catalysis and processivity. These critical interactions are transient in nature and remain largely uncharacterized. We have developed tools that allow synthetic modification of carrier protein domains, and here we leverage these tools to increase the lifetime of these protein-protein interactions using substrate mimics and covalent cross-linking inhibitors. These tools will allow us to visualize the molecular details by which carrier proteins bind tethered substrates prior to catalysis and how they interact with cognate catalytic domains. Through these studies, we intend to gain a more complete understanding of the dynamics of these catalytic events through analysis of solution phase NMR spectra. NMR perturbation comparisons of modified carrier proteins and titration experiments in the presence of catalytic domains will offer us an ability to accurately pinpoint the residues involved in substrate sequestration and protein-protein interaction. We will further validate our findings through site-directed mutagenesis, hydrogen/deuterium exchange NMR, and isothermal titration calorimetry. A deeper understanding of these protein-protein interactions will affect emerging fields in drug discovery. Using these tools to guide new interactions will enable combinatorial biosynthesis of new pharmacophores, while new drug targets may be discovered through abrogating these interactions in pathogen biosynthesis. PUBLIC HEALTH RELEVANCE: This program investigates the activity of modular synthases, a group of natural product biosynthetic enzymes of three types: polyketide synthase, non-ribosomal peptide synthase, and fatty acid synthase. These three subgroups are responsible for the production of antibiotics that represent major constituents of medicinal therapeutics, toxins that support pathogenesis for infection, and primary metabolites essential for life. These studies will benefit human health by enabling manipulation of these pathways to provide new therapeutic drugs and to identify new targets for drug design.